Figure 1.
Asymmetric proteasome distribution requires AIS integrity.(A and B) Either AnkG knockdown or induction of ischemia-like conditions (OGD) impairs AIS structure and proteasome distribution in cultures of mouse neurons on 5 DIV. Images of hippocampal neurons transfected with scrambled control (SC-siRNA) or AnkG-siRNA 30 min after plating, followed by immunostaining for the AIS marker AnkG (red), proteasome subunit Rpt5 (green, A), or the proteasome adaptor Ecm29 (green, B) at 5 DIV. Rightmost panels show regions of interest (dashed boxes) of the AIS represented at higher magnification, with Rpt5 (A) or Ecm29 (B) staining intensity indicated by a linear pseudocolor scale. Plots at right reveal a more uniform distribution (reflected by flatter slopes) of Rpt5 (blue trace, A) and Ecm29 (blue trace, B) across the AIS region and distal axon following AnkG knockdown or after 30 min of OGD conditions. Scale bar, 20 µm. Traces of neuron-specific class III β-tubulin (Tuj1) show that the microtubule bundle was not altered following AnkG knockdown. Traces represent averages of >50 cells per group from three independent experiments. m, slope of the intensity profile of Rpt5 or Ecm29 across the AIS. (C and D) MV151-labeled proteasomes are retained in the AIS region. (C) Top: Representative image showing a 5-DIV cortical neuron stained with neurofascin antibody and MV151 to visualize AIS position and proteasome movement, respectively, during live-cell imaging. Middle: Schematic showing position of the AIS (neurofascin-positive segment) and distal axon regions, defined as the axonal segment 60–80 µm from cell body. Bottom: Representative kymographs (2 s/frame; 180 s) of MV151-labeled proteasomes in axons of 5-DIV neurons following treatments indicated at left. Corresponding color-coded trajectories plotted for a subset of MV151-labeled proteasomes (blue lines, particles moving through AIS region; black lines, particles blocked or static in the proximal axon). Static particles outside the AIS are not marked. (D) Summary histograms showing that either AnkG knockdown by siRNA-AnkG or ischemia-like conditions (OGD for 30 min) significantly increase the percentage of MV151-labeled proteasomes across the AIS (denoted as “moving through”) in 5- or 6-DIV neurons. Note that treatment with the actin filament–depolymerizing agent latrunculin A (Lat A; 1 µM for 6 h) had no effect on proteasome transport, whereas the microtubule-disrupting agent nocodazole (Noco; 1 µM for 3 h) or Ecm29 KO (Ecm29−/−) abolished all transport. con, Control Ecm29+/+ group; #, no transport. Mean ± SEM, n = 20 cells per group from three independent experiments, 10–15 puncta each cell; *, P < 0.05; **, P < 0.01; ns, not significant compared with control group by unpaired t test. Scale bar, 20 µm. (E) Quantification of proteins at the AIS relative to those in the distal axon region in experiments conducted as described in C and D, except that 5-DIV cortical neurons were immunostained for Ecm29, Rpt5, and AnkG. Summary histograms show an even distribution of Ecm29 and Rpt5 over the AIS and distal axonal region in AnkG knockdown (AnkG-siRNA) neurons or OGD conditions, as indicated. Data represent mean ± SEM; n > 4 independent experiments, >50 cells per group; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant compared with control group by unpaired t test (Ecm29 and Rpt5 panels), or by one-way ANOVA with Dunnett’s multiple comparison test (AnkG panel).

Asymmetric proteasome distribution requires AIS integrity. (A and B) Either AnkG knockdown or induction of ischemia-like conditions (OGD) impairs AIS structure and proteasome distribution in cultures of mouse neurons on 5 DIV. Images of hippocampal neurons transfected with scrambled control (SC-siRNA) or AnkG-siRNA 30 min after plating, followed by immunostaining for the AIS marker AnkG (red), proteasome subunit Rpt5 (green, A), or the proteasome adaptor Ecm29 (green, B) at 5 DIV. Rightmost panels show regions of interest (dashed boxes) of the AIS represented at higher magnification, with Rpt5 (A) or Ecm29 (B) staining intensity indicated by a linear pseudocolor scale. Plots at right reveal a more uniform distribution (reflected by flatter slopes) of Rpt5 (blue trace, A) and Ecm29 (blue trace, B) across the AIS region and distal axon following AnkG knockdown or after 30 min of OGD conditions. Scale bar, 20 µm. Traces of neuron-specific class III β-tubulin (Tuj1) show that the microtubule bundle was not altered following AnkG knockdown. Traces represent averages of >50 cells per group from three independent experiments. m, slope of the intensity profile of Rpt5 or Ecm29 across the AIS. (C and D) MV151-labeled proteasomes are retained in the AIS region. (C) Top: Representative image showing a 5-DIV cortical neuron stained with neurofascin antibody and MV151 to visualize AIS position and proteasome movement, respectively, during live-cell imaging. Middle: Schematic showing position of the AIS (neurofascin-positive segment) and distal axon regions, defined as the axonal segment 60–80 µm from cell body. Bottom: Representative kymographs (2 s/frame; 180 s) of MV151-labeled proteasomes in axons of 5-DIV neurons following treatments indicated at left. Corresponding color-coded trajectories plotted for a subset of MV151-labeled proteasomes (blue lines, particles moving through AIS region; black lines, particles blocked or static in the proximal axon). Static particles outside the AIS are not marked. (D) Summary histograms showing that either AnkG knockdown by siRNA-AnkG or ischemia-like conditions (OGD for 30 min) significantly increase the percentage of MV151-labeled proteasomes across the AIS (denoted as “moving through”) in 5- or 6-DIV neurons. Note that treatment with the actin filament–depolymerizing agent latrunculin A (Lat A; 1 µM for 6 h) had no effect on proteasome transport, whereas the microtubule-disrupting agent nocodazole (Noco; 1 µM for 3 h) or Ecm29 KO (Ecm29−/−) abolished all transport. con, Control Ecm29+/+ group; #, no transport. Mean ± SEM, n = 20 cells per group from three independent experiments, 10–15 puncta each cell; *, P < 0.05; **, P < 0.01; ns, not significant compared with control group by unpaired t test. Scale bar, 20 µm. (E) Quantification of proteins at the AIS relative to those in the distal axon region in experiments conducted as described in C and D, except that 5-DIV cortical neurons were immunostained for Ecm29, Rpt5, and AnkG. Summary histograms show an even distribution of Ecm29 and Rpt5 over the AIS and distal axonal region in AnkG knockdown (AnkG-siRNA) neurons or OGD conditions, as indicated. Data represent mean ± SEM; n > 4 independent experiments, >50 cells per group; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant compared with control group by unpaired t test (Ecm29 and Rpt5 panels), or by one-way ANOVA with Dunnett’s multiple comparison test (AnkG panel).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal